Recently, the development of alternative energy to oil is a very important issue, because of the concern related to transportation energy supply, such as large increases in oil prices and the petroleum depletion prediction in the near future (peak oil), as well as environmental problems such as global warming and aerial pollution. Plant biomass, or lignocellulose, is the most plentiful renewable energy source on earth, which is expected to serve as an alternative source to oil. The main components in the dry weight of biomass are polysaccharides such as celluloses and hemicelluloses, and lignin. For example, polysaccharides are used as a biofuel or a raw material of chemical products, after being hydrolyzed into monosaccharides such as glucose or xylose by glycoside hydrolases which are collectively referred to as cellulase enzymes.
Lignocellulose is recalcitrant due to its highly complicated structures, and is hard to degrade with a single cellulolytic enzyme. Lignocellulose degradation to sugar requires at least three types of enzymes: endoglucanases (cellulase or endo-1,4-β-D-glucanase, EC 3.2.1.4) which randomly cut internal sites on cellulose chain, cellobiohydrolases (1,4-β-cellobiosidase or cellobiohydrolase, EC 3.2.1.91) which act as an exo-cellulase on the reducing or non-reducing ends of cellulose chain and release cellobiose as major products, and β-glucosidases (EC 3.2.1.21) which hydrolyze cellobiose to glucose. Besides, it is thought to be necessary to have an appropriate blending of a plurality of enzymes including xylanase (endo-1,4-β-xylanase, EC 3.2.1.8) which is a hemicellulase and other plant cell wall degrading enzymes.
In the lignocellulose to ethanol conversion process, high-solid loading up to 30-60% in initial substrate concentration has been attempted for the purpose of higher energy efficiency and less water usage. The enzymatic reaction in the high-solid loading processes is, however, hardly progressed because of high viscosity of slurries. It is clear that thermostable enzymes have an advantage to allow the use of increased substrate concentrations, because the substrate viscosity decreases as the temperature increases. Moreover, high temperatures generally accelerate catalytic reaction according to the van't Hoff Arrhenius law, and promote better enzyme penetration and cell-wall disorganization of the raw materials. Thus, if lignocellulose hydrolysis is processed at higher temperatures than the conventional temperature by using thermostable enzymes, more efficient biomass to sugar conversion will be achieved, resulting in largely cutting down of the enzyme amount and the time for hydrolysis so as to largely reduce the cost of the enzymes.
The temperature limit of living for thermophilic filamentous fungi, which are eukaryotic, is lower at about 55° C., than those of thermophilic bacteria and hyperthermophilic archaea, which are prokaryotic. For this reason, the thermostability of glycoside hydrolases expressed or secreted from thermophilic filamentous fungi is generally not so high. The filamentous fungus-derived CBH (cellobiohydrolases) so far reported to have the highest thermostability are cellobiohydrolases CBHI and CBHII from a thermophilic filamentous fungus Chaetomium thermophihum, respectively showing the optimum temperatures of 75° C. and 70° C. (for example, see Non-patent document 1), and cellobiohydrolase CBHI from Thermoascus aurantiacus showing the optimum temperature of 65° C. (for example, see Non-patent document 2). There is also a method to enhance the thermostability by substituting one or a plurality of amino acids in cellobiohydrolase (for example, see Patent Documents 1 or 2). However, the thermostability of mutated cellobiohydrolase obtained in such a manner is not yet sufficient.
On the other hand, thermophiles which proliferate in extreme environments such as hot springs, hydrothermal vents, oil fields, or metalliferous mines, at 55° C. or higher, or hyperthermophiles which proliferate at 80° C. or higher, have been isolated and cultured. The thermostable glycoside hydrolases derived from these thermophilic bacteria and hyperthermophilic archaea are mostly enzymes having endoglucanase activity, xylanase activity, xylosidase activity, or glucosidase activity. Regarding cellobiohydrolases which play the most important role in the lignocellulose hydrolysis process, there have been only several cellobiohydrolases isolated from three kinds of thermophilic bacteria belonging to the genus Clostridium, the genus Thermobifida, and the genus Thermotoga. For example, a thermophilic anaerobic bacterium Clostridium thermocellum, presents an enzyme complex termed cellulosome which has high lignocellulose hydrolysis activity, to outside the bacterial body. The main enzyme of the cellulosome is cellobiohydrolase, and three types thereof, namely. CelO belonging to the GH5 family, and CbhA and CelK belonging to the GH19 family, have been isolated. All of them have the optimum temperatures (Topt) of 60 to 65° C. (for example, see Non-patent documents 3 to 5). From a thermophilic actinobacterium Thermobifida fusca, there have been two different types of cellobiohydrolase genes isolated: E3 belonging to the GH6 family (for example, see Non-patent document 6), and Cel48A belonging to the GH48 family (for example, see Non-patent document 7). These cellobiohydrolases have relatively high thermostability. The temperature range at which they exhibit 50% activity of the maximum value is from 40 to 60° C. and a stable activity is held at 55° C. for at least 16 hours. However, these two types of cellobiohydrolases have insufficient activity at a temperature of 70° C. or higher. If an enzymatic hydrolysis process of cellulose is conducted using these two types of cellobiohydrolases, the upper limit temperature for the process would be 60 to 65° C. The cellobiohydrolase derived from a thermophilic bacterium belonging to the genus Thermotoga has the highest thermostability, and it has been reported to have the Topt of 105° C. and the activity half-life time (Thalf) of 70 minutes at 108° C. (for example, see Non-patent document 8). However, the enzyme shows an endoglucanase-like substrate specificity, and exhibits the degradation activity only to amorphous structured cellulose and carboxymethyl cellulose (CMC). Furthermore, because of a weak hydrolysis activity to a filter paper, efficient hydrolysis of crystalline lignocellulose cannot be expected with this enzyme.